Right ventricular (RV) contractile failure from acute pulmonary hypertension is an important cause of morbidity and mortality in conditions such as massive pulmonary embolism, hypoxic pulmonary vasoconstriction, and following cardiopulmonary bypass and cardiac transplantation, but effective therapeutic strategies are not currently available. Studies in the applicant's laboratory performed in a large animal model have demonstrated that RV contractile failure from acute pulmonary hypertension is associated with activation of the cysteine protease calpain in the RV, and that right heart failure in this setting may be attenuated by a calpain inhibitor. Calpain activation has been shown to occur in numerous models of both skeletal and cardiac muscle dysfunction, but there is controversy over both the upstream mechanism of calpain activation and the downstream targets of activated calpain: depending on the experimental model, contractile dysfunction has been attributed to disruption of structural proteins such as desmin, spectrin, 1-actinin or titin; modification of regulatory proteins such as troponin-I, troponin-T; or initiation of apoptosis. The overall goal of this project is to identify the mechanism by which acute RV pressure overload causes calpain activation and right ventricular contractile dysfunction during and following acute RV pressure overload. We have shown that degradation of the focal adhesion complex protein talin, a known calpain substrate, is highly correlated with the severity of RV dysfunction, and have obtained preliminary data showing that calpain redistributes to the myocyte membrane during acute pulmonary hypertension. Other investigators have proposed that calpain may be regulated by the non-voltage gated calcium channel TRPM7, by direct phosphorylation by the extracellular response kinase ERK, or by phosphorylation of calpain targets by the tyrosine kinase src. Therefore, we propose the following two hypotheses: Hypothesis #1: Acute pulmonary hypertension induced activation of calpain is dependent on the calcium channel TRPM7, ERK, and/or src. Hypothesis #2: Localized calpain activation in acute pulmonary hypertension causes RV dysfunction through degradation of focal adhesion complex (costameric) proteins such as talin. To test these hypotheses, we propose the following Specific Aims: Specific Aim 1: Employ a new ex vivo model of acute RV pressure overload using rodent RV papillary muscle to confirm findings from our established in vivo porcine model of acute RV pressure overload. Specific Aim 2: Identify signaling pathways and protein modifications that contribute to pressure overload induced RV dysfunction. Specific Aim 3: Determine if pressure overload induced RV dysfunction depends on TRPM7, ERK or src. Specific Aim 4: Determine whether protease or protein kinase inhibitors currently in clinical trials can attenuate right heart failure in acute RV pressure overload. We will use an established large animal (pig) in vivo model of acute pulmonary hypertension in conjunction with a new ex vivo model of acute RV pressure overload employing isolated rat RV papillary muscles. Rat RV papillary muscles subjected to contractile stress, modeling acute RV pressure overload, will be assessed for biochemical and histological evidence of protein redistribution and modification, and the effects of calpain and protein kinase inhibitors on these alterations will be determined. Parallel experiments performed in vivo in the pig model will be used to establish the physiological significance of findings from the ex vivo rodent papillary muscle model. Agents with potential clinical utility will be identified using the isolated papillary muscle model and tested in the pig model.